To identify targets of ubiquitin mediated protein degradation playing key roles in normal development, and that might potentially contribute to neoplasia when dysregulated, we carried out a yeast two hybrid screen for interaction partners of the E3 ubiquitin ligase Nedd4. This screen identified N4BP1 and N4BP3, two novel developmentally expressed proteins, each of which are now being characterized further because of the potential involvement of each in tumorigenesis. N4BP1 is a member of a small family of proteins containing a NYN motif, a domain predicted to have ribonuclease activity. N4BP1 undergoes polyubiquitination mediated by Nedd4, and also undergoes conjugation with SUMO, which in turn regulates N4BP1 ubiquitination and stability. Recent work has shown that N4BP1 also interacts with the related E3 ligase, ITCH, but is not a substrate for ITCH mediated ubiquitination. Rather, N4BP1 binding to ITCH, negatively regulates ITCH E3 activity directed toward its substrates, including the p53 related tumor suppressor proteins p73 and p63, as well as c-Jun. These results suggest that N4BP1 may have a role in regulating tumor progression and the response of cancer cells to chemotherapy. A potential role in tumorigenesis has recently come to light for the second Nedd4 interaction partner under study, N4BP3. The locus has been identified as a common site for retroviral integration in mouse leukemogenesis. In addition, N4BP3 is now known to contain a so-called FEZ domain first identified in the tumor suppressor, LZTS1. Our recent studies have shown that Smurf1 and Smurf2, E3 ubiquitin ligases related to Nedd4 and implicated in the regulation of TGF-&#946; and MEKK signaling, can mediate N4BP3 polyubiquitination and proteasomal degradation, suggesting a function for N4BP3 in these pathways. Finally, a yeast two-hybrid screen using N4BP3 as bait revealed an interaction with the SIPA1 family of proteins, implicated in metastasis. We validated this finding by demonstrating co-immunoprecipitation of N4BP3 and SIPA1, and are now carrying out a collaborative study to determine if this interaction has implications for SIPA1 function. Our studies on the SUMO pathway have as a primary focus the analysis of the desumoylating enzyme SENP1, and specifically its role in regulating cell proliferation. We previously showed that mutation of the Senp1 gene in mice leads to placental defects stemming from continued proliferation of trophoblast precursor cells at the expense of differentiation into mature placental labyrinth cells. Primary cell lines derived from SENP1 mutants also show an increased rate of proliferation as compared to wild type. Consistent with this finding, we observe an up-regulation of E2F target genes such as cdc2 and cyclinA2 in mutant cells and an increase in Retinoblastoma (RB) protein phosphorylation. Interestingly, RB recently was shown to be sumoylated, with the sumoylated form exerting less repressive potential on E2F activity. Our own analysis indicates that RB is a target SENP1 mediated desumoylation. Thus, we hypothesize that increased steady state sumoylation of RB may underlie the increased cellular proliferation seen in SENP1 mutant cells. We are currently assessing the ability of a mutant form of RB, which cannot be sumoylated, to rescue the cell cycle defects. In addition to these studies we are carrying out a proteomic scale mass spectrometry based analysis of SENP1 targets. Preliminary studies in primary embryonic fibroblasts have demonstrated the power of this approach to identify protein(s) whose increased steady state level of sumoylation potentially underlies the SENP1 mutant phenotype. We have also introduced specific sequence variations into SUMO that do not compromise conjugation activity but do facilitate mass spectrometric identification of the actual site of SUMO conjugation on target proteins, an essential step in allowing validation and in understanding the physiological role of sumoylation in individual protein function. Complementing our work on SENP1, we are investigating the consequences of genetic loss of SUMO during development. Surprisingly, we have found that mice with loss-of-function mutations in the SUMO-1 gene are normal. Further analysis has shown that there is compensatory utilization of the related SUMO-2/3 proteins, which apparently rescues the loss of SUMO-1. This finding has important implications for ongoing efforts to develop rational approaches to targeting the SUMO pathway, illustrating the need to take into account that these related SUMO molecules can substitute for each other